Fig. 8. Beclin 1 depletion in Molm-13 promotes necroptosis in vitro and in vivo.
Molm-13 cells expressing shGFP and shBECN1 #5 were treated with 0.05 μM birinapant and 20 μM Z-VAD-FMK (Biri/Z) (a) or 1 μM emricasan (Biri/Emri) (b) in the presence or absence of 100 nM GSK'963 for 10 h, and then stained with propidium iodide (PI) for 15 min. After staining, the cell death was determined by flow cytometry. The data are mean ± S.D., n = 3, with ***P < 0.001 at each point compared to the indicated graph using a two-sided Student’s t test. Molm-13 cells expressing indicated shRNAs were treated with 0.05 μM birinapant and 20 μM Z-VAD-FMK (Biri/Z) (c) or 1 μM emricasan (Biri/Emri) (d) in the presence or absence of 100 nM GSK'963 for 10 h. The cells were lysed under reducing conditions or under non-reducing conditions for MLKL oligomerisation, and analysed by immunoblotting. e Scheme for the subcutaneous tumour xenograft model. f–i 5 × 105 Molm-13 cells expressing indicated shRNAs were subcutaneously injected in the flank of 6-week-old female nude mice. After one week, the mice bearing tumours were injected with birinapant (2 mg/kg) plus emricasan (1 mg/kg) as indicated in (e) by i.p. injection for 2 weeks. Tumour growth (f), tumour-bearing mice (g), resected tumours (h), tumour masses (i) are shown. Data are means and individual data points from n = 6 mice, with *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. = non-significance according to the two-tailed Mann–Whitney test.